MULTI-PART OLIGONUCLEOTIDE PROBES AND METHODS OF USE THEREOF
Disclosed herein, inter alia, are oligonucleotides, methods, and kits useful for amplifying and detecting targets such as nucleic acids, proteins, and carbohydrates.
1 . A method of generating an amplification product, said method comprising:
a) contacting a target polynucleotide in a cell or tissue with a first oligonucleotide comprising a first target hybridization sequence, wherein said first oligonucleotide comprises a first probe hybridization sequence, a barcode sequence, and a second probe hybridization sequence, and hybridizing the first target hybridization sequence to a first target sequence of said target polynucleotide;
b) contacting the target polynucleotide with a second oligonucleotide comprising a second target hybridization sequence, wherein said second oligonucleotide comprises a third probe hybridization sequence and a fourth probe hybridization sequence, and hybridizing the second target hybridization sequence to a second target sequence of said target polynucleotide;
c) hybridizing a first oligonucleotide probe to said first oligonucleotide and to said second oligonucleotide, wherein said first oligonucleotide probe comprises a sequence complementary to said second probe hybridization sequence and a sequence complementary to said fourth probe hybridization sequence;
d) hybridizing a second oligonucleotide probe to said first oligonucleotide and said second oligonucleotide, wherein said second oligonucleotide probe comprises a sequence complementary to said first probe hybridization sequence and a sequence complementary to said third probe hybridization sequence;
e) extending the second oligonucleotide probe along the barcode sequence with a polymerase to generate a complementary barcode sequence, ligating the complementary barcode sequence to the first oligonucleotide probe, and ligating the first oligonucleotide probe to the second oligonucleotide probe, thereby generating a circular oligonucleotide; and
f) amplifying the circular oligonucleotide, thereby generating an amplification product comprising multiple complements of the complementary barcode sequence.
2 . The method of claim 1 , further comprising binding a plurality of oligonucleotides to the target polynucleotide and repeating steps c), d), e) and f) for each bound oligonucleotide.
3 . The method of claim 1 , further comprising detecting the amplification product of step (f).
4 . The method of claim 3 , wherein detecting the amplification product comprises hybridizing an oligonucleotide associated with a detectable label to the amplification product and identifying said detectable label.
4 . The method of claim 1 , further comprising sequencing the amplification product of step (f).
6 . The method of claim 5 , wherein sequencing comprises sequencing by synthesis, sequencing by binding, sequencing by ligation, or pyrosequencing.
7 . The method of claim 5 , wherein sequencing comprises extending a sequencing primer by incorporating a labeled nucleotide or labeled nucleotide analogue, and detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue, wherein the sequencing primer is hybridized to the extension product.
5 . The method of claim 1 , wherein steps (a)-(f) of the method are performed in a cell.
9 . The method of claim 8 , wherein the cell is permeabilized and immobilized to a solid support surface.
6 . The method of claim 1 , wherein said second oligonucleotide does not comprise a barcode sequence.
7 . The method of claim 1 , wherein said first oligonucleotide probe and second oligonucleotide probe do not comprise a barcode sequence.
8 . The method of claim 1 , wherein the first target sequence is 5′ to the second target sequence of said target polynucleotide.
9 . The method of claim 1 , wherein amplifying the circular oligonucleotide comprises incubating the circular oligonucleotide with the strand-displacing polymerase (a) for about 1 minute to about 2 hours, and/or (b) at a temperature of about 20° C. to about 50° C.
10 . The method of claim 1 , wherein the first oligonucleotide, the second oligonucleotide, or both the first and second oligonucleotides comprise one or more locked nucleic acid (LNA) nucleotides.
11 . The method of claim 1 , wherein the first oligonucleotide probe, the second oligonucleotide probe, or both the first and second oligonucleotide probes comprise one or more locked nucleic acid (LNA) nucleotides.
12 . The method of claim 1 , wherein the circular oligonucleotide is about 100 to about 1000 nucleotides in length.
13 . The method of claim 1 , wherein each target hybridization sequence is about 5 to about 35 nucleotides in length.
14 . The method of claim 1 , wherein the barcode sequence is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
15 . The method of claim 1 , wherein the barcode sequence is selected from a known set of barcode sequences.
20 . The method of claim 19 , wherein each of the known set of barcode sequences is associated with a target hybridization sequence from a known set of target hybridization sequences.